Error in ngs.plot.r

[Federico Gaiti]

Hi,

I installed ngs.plot as per instructions and install the Aqu1 supported genome. This step worked fine. 

When using ngs.plot.r I get this:

ngs.plot.r -G Aqu1 -R tss -C H3K4me3_PE_fixed_sorted.bam -O H3K4me3_PE.tss -T H3K4me3_PE -FL 300 -RB 0.05
Configuring variables...
Using database:
/hox/u/uqfgaiti/Tools/ngsplot/database/Aqu1/Aqu1.ensembl.genebody.protein_coding.RData
Done
Loading R libraries.....Done
Analyze bam files and calculate coverage.Error in { :
task 1 failed - "no applicable method for 'within' applied to an object of class "NULL""
Calls: covMatrix -> %dopar% -> <Anonymous>
Execution halted

I already changed the chromosome name in the header bam file using samtools reheader from Contig1000 to chrContin1000 for example. 

I re-installed R and the required packages and re-installed ngs.plot again. Still getting the same error. 

Any clue on what's going on?

Thanks

Federico

[Li Shen]

This error is being reported a lot recently. It seems to be related with the Rsamtools package that ngs.plot depends. Some user reported that its folder is locked and is not upgraded with R. So you need to make sure it is upgraded with your R installation and then try again.

Li

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Li Shen, Ph.D. Computer Science
Assistant Professor
Department of Neuroscience
Icahn School of Medicine at Mount Sinai
New York, NY 10029
#################
ngs.plot - the coolest NGS plotting tool: https://code.google.com/p/ngsplot/

Want ChIP-seq differential analysis? Use: https://code.google.com/p/diffreps/

[Federico Gaiti]

Hi Li,

so I tried to re-instelled Rsamtools (this should install the latest version I guess) and this is what I get at the end: 

* R

** inst

** preparing package for lazy loading

** help

*** installing help indices

** building package indices

** installing vignettes

** testing if installed package can be loaded

* DONE (Rsamtools)

The downloaded source packages are in

‘/tmp/Rtmp58C4Ln/downloaded_packages’

Warning message:

installed directory not writable, cannot update packages 'boot', 'class',

  'cluster', 'codetools', 'KernSmooth', 'MASS', 'mgcv', ‘nlme'

It seems I have the same problem as previous users, some packages cannot be updated. Do you think that is this the problem why ngs.plot.r is not working? 

Sorry I have really limited experience in bioinformatics.

Thanks for help

Federico

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[Li Shen]

Those packages are installed under system directory and you need administrative privilege to upgrade them. But I doubt it is related. Have you tried ngs.plot again?

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[Federico Gaiti]

Yes. Got this again:

ngs.plot.r -G Aqu1 -R tss -C H3K4me3_PE_fixed_sorted.bam -O H3K4me3_PE.tss -T H3K4me3_PE -FL 300 -RB 0.05

Configuring variables...

Using database:

/hox/u/uqfgaiti/Tools/ngsplot/database/Aqu1/Aqu1.ensembl.genebody.protein_coding.RData

Done

Loading R libraries.....Done

Analyze bam files and calculate coverage.Error in { :

  task 1 failed - "no applicable method for 'within' applied to an object of class "NULL""

Calls: covMatrix -> %dopar% -> <Anonymous>

Execution halted

Federico

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[pranitha vangala]

Hi Li

I too get the same error as federico. Below is my command and error

ngs.plot.r -G hg19 -R bed -E ~/Desktop/xxx.bed-C ~/Desktop/input.bam -O output_ngs -RB 0.05 -P 1

Configuring variables...Done

Loading R libraries.....Done

Analyze bam files and calculate coverage..................................................................................................................................................................................................Error in { : 

  task 1 failed - "no applicable method for 'within' applied to an object of class "NULL""

Calls: covMatrix -> %dopar% -> <Anonymous>

Execution halted

[ningyi shao]

Hi Federico and Pranitha,

Would you mind telling us the version of R, ngs.plot, and Rsamtools?

Bests,

Ning-Yi

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[pranitha vangala]

Hi Ning-Yi

Im using R 3.1.1,ngs.plot 2.41.3, Rsamtools 1.18.1

[Li Shen]

Could this be due to the newest version of Rsamtools? This is my R session info:

> sessionInfo()

R version 3.1.1 (2014-07-10)

Platform: x86_64-pc-linux-gnu (64-bit)

locale:

 [1] LC_CTYPE=en_US.UTF-8       LC_NUMERIC=C              

 [3] LC_TIME=en_US.UTF-8        LC_COLLATE=en_US.UTF-8    

 [5] LC_MONETARY=en_US.UTF-8    LC_MESSAGES=en_US.UTF-8   

 [7] LC_PAPER=en_US.UTF-8       LC_NAME=C                 

 [9] LC_ADDRESS=C               LC_TELEPHONE=C            

[11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C       

attached base packages:

[1] parallel  stats     graphics  grDevices utils     datasets  methods  

[8] base     

other attached packages:

[1] Rsamtools_1.16.1     Biostrings_2.32.0    XVector_0.4.0       

[4] GenomicRanges_1.16.3 GenomeInfoDb_1.0.2   IRanges_1.22.9      

[7] BiocGenerics_0.10.0 

loaded via a namespace (and not attached):

[1] bitops_1.0-6    stats4_3.1.1    zlibbioc_1.10.0

my Rsamtools is older than yours.

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[pranitha vangala]

Im not sure how to install the same version Rsamtools. When i try i get the below error

package ‘Rsamtools_1.16.1’ is not available (for R version 3.1.1) 

[Federico Gaiti]

Hi,

I’m too using R 3.1.1, ngs.plot 2.41.3 and Rsamtools 1.18.

Thanks

Federico

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[Federico Gaiti]

Do you have any suggestion on how to install an older version of Rsamtools than 1.18.1? 

Thanks

Federico

On 1 Nov 2014, at 06:01, pranitha vangala <pranitha...@gmail.com> wrote:

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[Yu Zhou]

Hi,

I now have the same problem as in this thread, after upgrading several packages.

Configuring variables...

Using database:

/home/y6zhou/soft/bio/NGSplot/ngsplot/database/hg18/hg18.refseq.genebody.protein_coding.RData

Done

Loading R libraries.....Done

Analyze bam files and calculate coverage............................................................................................................................................................................................Error in { : 

  task 1 failed - "no applicable method for 'within' applied to an object of class "NULL""

Calls: covMatrix -> %dopar% -> <Anonymous>

Execution halted

R3.1.1, Rsamtools_1.18.1 

Any suggestion to solve this problem? Thanks a lot!

Best,

Yu

[Federico Gaiti]

I tried again to re-install everything from scratch but still getting the same error. If the problem might be in Rsamtools 1.18.1., does anyone know how to install a precedent version? It seems that version 1.18.1 get installed automatically when using Bioconductor 3.0.

Thanks for help

Federico

[Li Shen]

Hi all, 

The problem has now been solved. It is caused by some recent upgrade in Bioconductor packages that changed the chromosome names in their internal data structures. 

Download the 2.41.4 version to fix the problem.

- Li

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[Federico Gaiti]

Thanks a lot Li. 

I tested it and it’s now working. I was able to make all the average profile plots for my marks. Though, do you have any idea why the heat map doesn’t work? I have attached the pdf here. 

My command was: ngs.plot.r -G Aqu1 -R genebody -C H3K4me3_SE_October_fixed_sorted.bam -O H3K4me3_SE_October.genebody -T H3K4me3_SE_October -FL 250 -RB 0.05

Thanks a lot for all the help

Federico

[Li Shen]

Your heatmap is fine. There are just very few genes. How many genes are there in this "Aqu1" genome? Can you check? There should be a program message indicating the R data file that is being used.

Li

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[Federico Gaiti]

Hi Li,

the Aqu1 has around 30000 genes. It seems weird that only very few genes show that histone marks. 

I can see the Rdata file but is there a way I could check how many genes have been used to generate the profile/heatmap?

Thanks for help

Federico

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[Li Shen]

Of course, ngs.plot produces a zip file. Unzip it and you'll find an R data file that contains the coverage matrix.

Is it possible that your bam file's chromosome names are not compatible with the ngs.plot database. Use "ngsplotdb.py chrnames Aqu1" to list the chromosome names in the database.

Li

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[Federico Gaiti]

Thanks. I previously used samtools reheader to modify the header of the bam files. They should match.

When I run the command you suggest, I got this:

ngsplotdb.py chrnames Aqu1

usage: ngsplotdb.py chrnames [-h] gn db

ngsplotdb.py chrnames: error: too few arguments

Am I missing anything? 

 

thanks

Federico

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[Li Shen]

ngsplotdb.py chrnames Aqu1 ensembl (or refseq).

Li

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[Federico Gaiti]

ngsplotdb.py chrnames Aqu1 ensembl 

chrContig100

chrContig10001

chrContig10002

chrContig10003

chrContig10005

chrContig10006

chrContig10007

chrContig10008

chrContig10009

chrContig1001

samtools view -H H3K4me3_SE_October_fixed_sorted.bam 

@HD VN:1.0 SO:unsorted

@SQ SN:chrContig1 LN:633

@SQ SN:chrContig3 LN:652

@SQ SN:chrContig4 LN:654

@SQ SN:chrContig5 LN:662

@SQ SN:chrContig6 LN:665

@SQ SN:chrContig7 LN:715

@SQ SN:chrContig8 LN:717

@SQ SN:chrContig9 LN:718

@SQ SN:chrContig10 LN:720

The chromosome names seem to match. It is really odd that only really few genes are present in the heat map…

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[Federico Gaiti]

Hi Li,

would have any other idea about why I would have so few genes in the heat map? 

My bam file’s chromosome names correspond with the nags.plot database and when I look at the peaks called by MACS2 on a genome browser I see a lot of genes showing that histone marks around the promoter region. 

Thanks for help

Federico

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